Viral hemorrhagic fevers caused by viruses belonging to the genus Ebolavirus and Marburgvirus, both members of the Filoviridae family, are among the most severe infectious diseases in human and nonhuman primates (NHPs), and no licensed vaccines or effective therapeutics are currently available. Ebola virus (EBOV), in particular, has been responsible for multiple Ebola hemorrhagic fever (EHF) outbreaks with case-fatality rates ranging from 65 to 90%. Studies with animal models and limited clinical data from EHF outbreaks suggest that interdependent pathogenic processes, including both the host immune and pathophysiological responses, induced by EBOV infection trigger the severe hemorrhagic syndrome. In order to develop effective treatments for EHF, it is necessary to better understand the mechanisms of viral and host interactions at the molecular and cellular levels and how these interactions contribute to the in vivo pathogenic process. Our research for this year is therefore focused on elucidating the functions of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have four ongoing projects: (1) characterization of the pathogenic processes in the Syrian hamster model that recapitulates EHF and (2) characterization of EBOV protein interactions. (1) Characterization of pathogenic processes in the Syrian hamster model, which recapitulates EHF. While the NHP model is used to evaluate the efficacy of EBOV vaccines and therapeutics because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of EBOV research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of EHF. Recently, we have developed and characterized a novel lethal Syrian hamster model of EHF based on infection with mouse-adapted EBOV that manifests many of the clinical and pathological findings observed in EBOV-infected NHPs and humans, including coagulation abnormalities. To determine the mechanisms of pathogenesis in this model, we have started to develop the tools for monitoring the host immune responses in infected animals. As an initial step, we performed Syrian hamster transcriptome sequencing for a cDNA library generated from pooled RNA isolated from the major organs of Syrian hamsters. The sequence reads were assembled into larger non-overlapping contigs (sequences), and expressed sequence tags (ESTs) were annotated based on other rodent species transcriptomes. We identified the most highly covered and highly expressed transcripts in our library and performed a functional enrichment analysis to identify which biological functions and canonical signaling pathways were significantly represented. This EST library improves our understanding of the Syrian hamster transcriptome, especially in the context of emerging infectious diseases, including Ebola hemorrhagic fever, hantavirus pulmonary syndrome, and henipavirus infections. Moreover, this library is a significant resource for the wider biomedical research community to help improve genome annotation of the Syrian hamster and closely related species. Finally, these data provide the basis for development of microarrays that can be utilized in functional genomics studies to understand the pathogenesis of Ebola hemorrhagic fever. (2) Characterization of EBOV protein interactions. Relatively little information exists regarding the molecular details that govern interactions between EBOV proteins. As such, we are actively interested in understanding the determinants of EBOV protein interaction and the functional outcomes of those interactions. The EBOV nucleoprotein (NP) and viral protein (VP) 24, both constituents of the viral nucleocapsid, are the sole factors responsible for EBOV virulence in mice, suggesting that these two proteins play a critical role in the induction of disease. Given their contribution to EBOV virulence, we sought to characterize the physical relationship between NP and VP24. We used confocal microscopy and immunoprecipitations to demonstrate that wild-type NP both co-localizes and interacts with VP24. A series of NP deletion mutants were then constructed to refine the determinants of this interaction. Ultimately, the N-terminus of NP was identified as the region necessary and sufficient for interaction with VP24. To determine the region on VP24 responsible for the interaction with NP, we performed bioinformatics analysis to identify the amino acids most likely to be involved in protein-protein interactions. Based on this prediction, we generated a series of VP24 mutants each with up to eight consecutive amino acids mutated to alanines throughout the protein. Assessing these mutants for their ability to interact with NP revealed a region near the C-terminus of VP24 that likely plays a critical role in the ability of VP24 to interact with NP. The work to elucidate the physical relationship between NP and VP24 has laid the foundation for understanding the functional relationship between these two proteins. Indeed, based on this and other work, we hypothesize that VP24 plays a critical role in condensing the nucleocapsid, thereby restricting viral replication and transcription and promoting nucleocapsid packaging and egress. Future work will focus on validating this hypothesis using a variety of techniques, including electron microscopy, reverse genetics, and minigenome or transcription/replication competent virus like particle systems.

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Banadyga, Logan; Dolan, Michael A; Ebihara, Hideki (2016) Rodent-Adapted Filoviruses and the Molecular Basis of Pathogenesis. J Mol Biol 428:3449-66
Tsuda, Yoshimi; Hoenen, Thomas; Banadyga, Logan et al. (2015) An Improved Reverse Genetics System to Overcome Cell-Type-Dependent Ebola Virus Genome Plasticity. J Infect Dis :
Hoenen, T; Safronetz, D; Groseth, A et al. (2015) Virology. Mutation rate and genotype variation of Ebola virus from Mali case sequences. Science 348:117-9
Cross, Robert W; Fenton, Karla A; Geisbert, Joan B et al. (2015) Comparison of the Pathogenesis of the Angola and Ravn Strains of Marburg Virus in the Outbred Guinea Pig Model. J Infect Dis :
Gabriel, Gülsah; Feldmann, Friederike; Reimer, Rudolph et al. (2015) Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice. J Infect Dis :
Pinto, Amelia K; Williams, Graham D; Szretter, Kristy J et al. (2015) Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families. J Virol 89:9465-76
Dunham, Eric C; Banadyga, Logan; Groseth, Allison et al. (2015) Assessing the contribution of interferon antagonism to the virulence of West African Ebola viruses. Nat Commun 6:8000
Hoenen, Thomas; Marzi, Andrea; Scott, Dana P et al. (2015) Soluble Glycoprotein Is Not Required for Ebola Virus Virulence in Guinea Pigs. J Infect Dis :
Wong, Gary; Qiu, Xiangguo; Ebihara, Hideki et al. (2015) Characterization of a Bivalent Vaccine Capable of Inducing Protection Against Both Ebola and Cross-clade H5N1 Influenza in Mice. J Infect Dis :
Hoenen, T; Groseth, A; Feldmann, F et al. (2014) Complete genome sequences of three ebola virus isolates from the 2014 outbreak in west Africa. Genome Announc 2:

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